Designing the optimum peptide antigen with the right antigenicity is difficult for the development of anti-peptide antibodies. Many parameters regarding the antigen peptide need to be optimized. They cover the peptides' purity levels, amino acid compositions, lengths, hydrophobicity, and secondary structures. In addition, the designing also considers difficult amino acids either for their avoidance or for special treatments. One of the axioms that GenScript respects is “Quality by Design”. Using OptimumAntigen Design Tool, GenScript's peptide designing process is in line with this particular motto, delivering quality peptide and antibody projects smartly designed right from the beginning.
One important advantage for synthetic peptides to work as antigens is that they be designed to cover a specific epitope of interest. Using advanced algorithms, peptide antigen candidates are screened against a specific protein databank to optimize their cross-reactivity and overall antibody specificity. Sequences with minimum homology are selected to reduce non-specific bindings.
Helped with GenScript's proprietary FlexPeptide technology, peptide antigens designed, synthesized, and conjugated by GenScript achieve a success rate of 85-90% when working as antigens delivering anticipated positive immune responses.
Shown below is a case study of using peptide antigen designed by OptimumAntigen Tool
For antibody generation and testing, peptide purity > 70% is enough, however, for biological activity studies, peptide purity > 95% is required. GenScript is able to develop peptides with different purity levels and has the ability to synthesize peptides with purity greater than 98%.
Peptide Amino Acid Composition
Amino acid composition governs every aspect of the peptide’s functionality. Peptide antigens should be designed to contain sequences that are found on the surface of the native protein. Both hydrophobic and hydrophilic residues should be included. In particular, it is favorable for the peptide sequence to incorporate amino acids that are antigenic.
On the other hand, GenScript peptide design process avoids problematic amino acids. In particular, cysteine, methionine, and tryptophan are susceptible to oxidation and side reactions. Peptides containing multiple instances of these residues are also difficult to be obtained in high purity. Sometimes, the peptides are chemically modified or conjugated with specific ligands for desired properties, such as solubility, stability, even purification methods. Additional modifications such as N- and C-termini capping, spacer insertions and amino acid replacement are also performed.
Peptide length is a critical factor to consider when synthesizing peptide. The longer the peptide sequence, the more difficult for their synthesis and purification. Longer peptides generally have lower crude peptide purity. For long and complex peptides, GenScript implements its proprietary recombinant peptide system and has the ability to produce peptides with lengths up to 200 residues.
GenScript recommends peptide antigens'length to cover 10-15 residues for antibody production.
Hydrophobicity of each constituted amino acids strongly influences the corresponding peptide’s solubility. By changing the frequency of occurrence of hydrophobic and hydrophilic amino acids, GenScript designs peptides of varied solubility. Peptides with high incidences of hydrophobic amino acids including tryptophan, leucine, valine, methionine, phenylalanine, and isoleucine may not easily dissolve in aqueous solutions. Hydrophobic amino acid content is suggested to be kept below 50%.At least one charged residue should be incorporated within every five amino acids. At physiological pH, arginine, glutamine, aspartic acid, and lysine all have charged side chains. A single conservative replacement or addition of polar residues to the N- or C-terminus may also improve peptide solubility.
During peptide synthesis, beta sheet formation can cause incomplete solvation of the growing peptide, which results in a high incidence of deletion sequences in the final product. GenScript’s peptide design process effectively eliminates this from happening. In general, GenScript sequences avoid multiple or adjacent residues of valine, isoleucine, tyrosine, phenylalanine, tryptophan, leucine, glutamine, or threonine to prevent beta sheet formation. If the above recommendation cannot be implemented practically, conservative replacement may help by reinserting a glycine or proline at every third residue or replacing glutamine with asparagine, or threonine with serine.
All peptide synthesis is subject to GenScript's stringent quality control. The typical delivery consists of lyophilized peptide of the required sequence, purity, and quantity and associated QC reports.